Chimeric insecticidal proteins toxic or inhibitory to lepidopteran pests

ABSTRACT

Nucleotide sequences are disclosed that encode novel chimeric insecticidal proteins exhibiting Lepidopteran inhibitory activity. Particular embodiments provide compositions and transformed plants, plant parts, and seeds containing the recombinant nucleic acid molecules encoding one or more of the chimeric insecticidal proteins.

REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.14/884,469, filed Oct. 15, 2015, which claims the benefit of U.S.provisional application No. 62/064,989, filed Oct. 16, 2014, each ofwhich are herein incorporated by reference in their entireties.

INCORPORATION OF SEQUENCE LISTING

A computer readable form of the Sequence Listing is filed with thisapplication by electronic submission and is incorporated into thisapplication by reference in its entirety. The Sequence Listing iscontained in the file created on Dec. 11, 2017, having the file nameP34230US03_SEQ.txt, and which is 371,792 bytes in size (as measured inthe MS-Windows® operating system).

FIELD OF THE INVENTION

The invention generally relates to the field of insect inhibitoryproteins. A novel class of chimeric insecticidal proteins exhibitinginsect inhibitory activity against agriculturally-relevant pests of cropplants and seeds is disclosed in this application. In particular, thedisclosed class of proteins exhibits insecticidal activity against theLepidopteran order of insect pests. Plants, plant parts, and seedscontaining a recombinant nucleic acid molecule encoding one or more ofthe disclosed toxin proteins are provided.

BACKGROUND OF THE INVENTION

Improving crop yield from agriculturally-significant plants including,among others, corn, soybean, sugarcane, rice, wheat, vegetables, andcotton, has become increasingly important. In addition to the growingneed for agricultural products to feed, clothe and provide energy for agrowing human population, climate-related effects and pressure from thegrowing population to use land other than for agricultural practices arepredicted to reduce the amount of arable land available for farming.These factors have led to grim forecasts with respect to food security,particularly in the absence of major improvements in plant biotechnologyand agronomic practices. In light of these pressures, environmentallysustainable improvements in technology, agricultural techniques, andpest management are vital tools to expand crop production on the limitedamount of arable land available for farming.

Insects, particularly insects within the order Lepidoptera, areconsidered a major cause of damage to field crops, thereby decreasingcrop yields in infested areas. Lepidopteran pest species whichnegatively impact agriculture include, but are not limited to, fallarmyworm (Spodoptera frugiperda), beet armyworm (Spodoptera exigua),bertha armyworm (Mamestra configurata), black cutworm (Agrotis ipsilon),cabbage looper (Trichoplusia ni), soybean looper (Chrysodeixisincludens), velvetbean caterpillar (Anticarsia gemmatalis), greencloverworm (Hypena scabra), tobacco budworm (Heliothis virescens),granulate cutworm (Agrotis subterranea), armyworm (Pseudaletiaunipuncta), western cutworm (Agrotis orthogonia), European corn borer(Ostrinia nubilalis), navel orangeworm (Amyelois transitella), corn rootwebworm (Crambus caliginosellus), sod webworm (Herpetogrammalicarsisalis), sunflower moth (Homoeosoma electellum), lesser cornstalkborer (Elasmopalpus lignosellus), codling moth (Cydia pomonella), grapeberry moth (Endopiza viteana), oriental fruit moth (Grapholita molesta),sunflower bud moth (Suleima helianthana), diamondback moth (Plutellaxylostella), pink bollworm (Pectinophora gossypiella), pink stem borer(Sesamia inferens), gypsy moth (Lymantria dispar), cotton leaf worm(Alabama argillacea), fruit tree leaf roller (Archips argyrospila),European leafroller (Archips rosana), Asiatic rice borer, or rice stemborer (Chilo suppressalis), rice leaf roller (Cnaphalocrocis medinalis),corn root webworm (Crambus caliginosellus), bluegrass webworm (Crambusteterrellus), southwestern corn borer (Diatraea grandiosella)),surgarcane borer (Diatraea saccharalis), spiny bollworm (Eariasinsulana), spotted bollworm (Earias vittella), Old World cotton bollworm(Helicoverpa armigera), corn earworm, soy podworm or cotton bollworm(Helicoverpa zea), sod webworm (Herpetogramma licarsisalis), Europeangrape vine moth (Lobesia botrana), citrus leafminer (Phyllocnistiscitrella), large white butterfly (Pieris brassicae), importedcabbageworm, or small white butterfly (Pieris rapae), tobacco cutworm,or cluster caterpillar (Spodoptera litura), and tomato leafminer (Tutaabsoluta).

Historically, the intensive application of synthetic chemicalinsecticides was relied upon as the pest control agent in agriculture.Concerns for the environment and human health, in addition to emergingresistance issues, stimulated the research and development of biologicalpesticides. This research effort led to the progressive discovery anduse of various entomopathogenic microbial species, including bacteria.

The biological control paradigm shifted when the potential ofentomopathogenic bacteria, especially bacteria belonging to the genusBacillus, was discovered and developed as a biological pest controlagent. Strains of the bacterium Bacillus thuringiensis (Bt) have beenused as a source for insecticidal proteins since it was discovered thatBt strains show a high toxicity against specific insects. Bt strains areknown to produce delta-endotoxins that are localized within parasporalcrystalline inclusion bodies at the onset of sporulation and during thestationary growth phase (e.g., Cry proteins), and are also known toproduce secreted insecticidal protein. Upon ingestion by a susceptibleinsect, delta-endotoxins as well as secreted toxins exert their effectsat the surface of the midgut epithelium, disrupting the cell membrane,leading to cell disruption and death. Genes encoding insecticidalproteins have also been identified in bacterial species other than Bt,including other Bacillus and a diversity of other bacterial species,such as Brevibacillus laterosporus, Lysinibacillus sphaericus (“Ls”formerly known as Bacillus sphaericus) and Paenibacillus popilliae.

Crystalline and secreted soluble insecticidal protein toxins are highlyspecific for their hosts and have gained worldwide acceptance asalternatives to chemical insecticides. For example, insecticidal toxinproteins have been employed in various agricultural applications toprotect agriculturally important plants from insect infestations,decrease the need for chemical pesticide applications, and increaseyields. Insecticidal toxin proteins are used to controlagriculturally-relevant pests of crop plants by mechanical methods, suchas spraying to disperse microbial formulations containing variousbacteria strains onto plant surfaces, and by using genetictransformation techniques to produce transgenic plants and seedsexpressing insecticidal toxin protein.

The use of transgenic plants expressing insecticidal proteins has beenglobally adopted. For example, in 2012, 26.1 million hectares wereplanted with transgenic crops expressing Bt toxins (James, C., GlobalStatus of Commercialized Biotech/GM Crops: 2012. ISAAA Brief No. 44).The global use of transgenic insect-protected crops and the limitednumber of insecticidal proteins used in these crops has created aselection pressure for existing insect alleles that impart resistance tothe currently-utilized insecticidal proteins.

The development of resistance in target pests to insecticidal proteinscreates the continuing need for discovery and development of new formsof insecticidal proteins that are useful for managing the increase ininsect resistance to transgenic crops expressing insecticidal proteins.New insecticidal proteins with improved efficacy and which exhibitcontrol over a broader spectrum of susceptible insect species willreduce the number of surviving insects which can develop resistancealleles. In addition, the use in one plant of two or more transgenicinsecticidal proteins toxic to the same insect pest and displayingdifferent modes of action reduces the probability of resistance in anysingle target insect species.

Consequently, there is a critical need to identify additionalinsecticidal proteins with improved insecticidal properties such asincreased efficacy against a broader spectrum of target insect pestsspecies and different modes of action compared to the toxins currentlyused in agricultural practices. To meet this need, the present inventiondiscloses novel Cry1 chimeric insecticidal proteins that exhibitactivity against significant target Lepidopteran pest species.

Members of the family of Cry1 crystal proteins are known in the art toexhibit bioactivity against Lepidopteran pests. The precursor form ofCry 1 crystal proteins consists of two approximately equal-sizedsegments. The carboxy-terminal portion of the precursor protein, knownas the protoxin segment, stabilizes crystal formation and exhibits noinsecticidal activity. The amino-terminal half of the precursor proteincomprises the toxin segment of the Cry1 protein and, based on alignmentof conserved or substantially conserved sequences within Cry1 familymembers, can be further sub-divided into three structural domains,domain I, domain II, and domain III. Domain I comprises about the firstthird of the active toxin segment and has been shown to be essential forchannel formation. Domains II and III have both been implicated inreceptor binding and insect species specificity, depending on the insectand insecticidal protein being examined.

The likelihood of arbitrarily creating a chimeric protein with enhancedproperties from the assortment of the domain structures of the numerousnative insecticidal proteins known in the art is remote. This is aresult of the complex nature of protein structure, oligomerization, andactivation (including correct proteolytic processing of the chimericprecursor, if expressed in such a form) required to release aninsecticidal protein segment. Only by careful selection of protoxins andspecific targets within each parental protein for the creation of achimeric structure can functional chimeric insecticidal toxins beconstructed that exhibit improved insecticidal activity in comparison tothe parental proteins from which the chimeras are derived. It is knownin the art that reassembly of the protoxin and toxin domains I, II andIII of any two or more toxins that are different from each other oftenresults in the construction of proteins that exhibit faulty crystalformation or the complete lack of any detectable insecticidal activitydirected to a preferred target insect pest species. Only by trial anderror are effective insecticidal chimeras designed, and even then, theskilled artisan is not certain to end up with a chimera that exhibitsinsecticidal activity that is equivalent to or improved in comparison toany single parental toxin protein from which the constituent protoxin ortoxin domains of the chimera may have been derived. For example, theliterature reports numerous examples of the construction or assembly ofchimeric proteins from two or more crystal protein precursors. See, e.g.Jacqueline S. Knight, et al. “A Strategy for Shuffling Numerous Bacillusthuringiensis Crystal Protein Domains.” J. Economic Entomology, 97 (6)(2004): 1805-1813; Bosch, et al. (U.S. Pat. No. 6,204,246); Malvar andGilmer (U.S. Pat. No. 6,017,534). In each of these examples, many of theresultant chimeras failed to exhibit insecticidal or crystal formingproperties that were equivalent to or improved in comparison to theprecursor proteins from which the components of the chimeras werederived.

SUMMARY OF THE INVENTION

Recombinant nucleic acid molecules are provided that encode chimericinsecticidal proteins toxic to Lepidopteran species of plant pests. Eachof the chimeric insecticidal proteins can be used alone or incombination with each other and with other insecticidal proteins andinsect inhibitory agents in formulations and in planta; thus providingalternatives to insecticidal proteins and insecticidal chemistriescurrently in use in agricultural systems.

In certain embodiments disclosed herein is a chimeric insecticidalprotein comprising an amino acid sequence as set forth in any of SEQ IDNOs: 21, 10, 28, 7, 4, 13, 16, 19, 23, 25, 30, 33, 36, 39, 41, 43, 45,47, 50 or 53. This chimeric insecticidal protein exhibits inhibitoryactivity against an insect species of the order Lepidoptera such as, butnot limited to, Anticarsia gemmatalis, Diatraea saccharalis,Elasmopalpus lignosellus, Helicoverpa zea, Heliothis virescens,Chrysodeixis includens, Spodoptera cosmioides, Spodoptera eridania,Spodoptera frugiperda, Spodoptera exigua, Helicoverpa armigera,Spodoptera litura, Pectinophora gossypiella, Diatraea grandiosella,Earias vitella, Helicoverpa gelotopeon, and Rachiplusia nu.

In another embodiment, a polynucleotide encoding a chimeric insecticidalprotein, wherein the polynucleotide is operably linked to a heterologouspromoter and the chimeric insecticidal protein comprises the amino acidsequence as set forth in any of SEQ ID NOs: 21, 10, 28, 7, 4, 13, 16,19, 23, 25, 30, 33, 36, 39, 41, 43, 45, 47, 50 or 53 is disclosed. Apolynucleotide encoding a chimeric insecticidal protein, wherein thepolynucleotide comprises a nucleotide sequence that optionally:hybridizes under stringent conditions to the reverse complement of thepolynucleotide sequence as set forth in any of SEQ ID NOs:1, 2, 3, 5, 6,8, 9, 11, 12, 14, 15, 17, 18, 20, 22, 24, 26, 27, 29, 31, 32, 34, 35,37, 38, 40, 42, 44, 46, 48, 49, 51 or 52; or encodes the chimericinsecticidal protein comprising an amino acid sequence as set forth inany of SEQ ID NOs: 21, 10, 28, 7, 4, 13, 16, 19, 23, 25, 30, 33, 36, 39,41, 43, 45, 47, 50 or 53 is also contemplated.

In other embodiments disclosed herein is a host cell comprising thepolynucleotide set forth in any of SEQ ID NO: 1, 2, 3, 5, 6, 8, 9, 11,12, 14, 15, 17, 18, 20, 22, 24, 26, 27, 29, 31, 32, 34, 35, 37, 38, 40,42, 44, 46, 48, 49, 51 or 52, wherein the host cell is selected from thegroup consisting of a bacterial host cell or a plant host cell.Contemplated bacterial host include Agrobacterium, Rhizobium, Bacillus,Brevibacillus, Escherichia, Pseudomonas, Klebsiella, and Erwinia; andwherein the Bacillus species is a Bacillus cereus or a Bacillusthuringiensis, said Brevibacillus is a Brevibacillus laterosperous, andsaid Escherichia is an Escherichia coli. Contemplated plant cellsinclude monocots and dicots.

Other embodiments disclosed herein include insect inhibitorycompositions comprising a chimeric insecticidal protein comprising anamino acid sequence as set forth in any of SEQ ID NOs: 21, 10, 28, 7, 4,13, 16, 19, 23, 25, 30, 33, 36, 39, 41, 43, 45, 47, 50 or 53. In certainembodiments, the insect inhibitory composition further comprises atleast one insect inhibitory agent different from the chimericinsecticidal protein. Contemplated insect inhibitory agents differentfrom the chimeric insecticidal protein include an insect inhibitoryprotein, an insect inhibitory dsRNA molecule, and an insect inhibitorychemistry. These insect inhibitory agents different from the chimericinsecticidal protein can exhibit activity against one or more pestspecies of the orders Lepidoptera, Coleoptera, Hemiptera, Homoptera, orThysanoptera.

In yet another embodiment, disclosed herein is a seed comprising aninsect inhibitory effective amount of: a chimeric insecticidal proteincomprising the amino acid sequence as set forth in any of SEQ ID NOs:21, 10, 28, 7, 4, 13, 16, 19, 23, 25, 30, 33, 36, 39, 41, 43, 45, 47, 50or 53; or a polynucleotide set forth in any of SEQ ID NOs:1, 2, 3, 5, 6,8, 9, 11, 12, 14, 15, 17, 18, 20, 22, 24, 26, 27, 29, 31, 32, 34, 35,37, 38, 40, 42, 44, 46, 48, 49, 51 or 52.

Methods of controlling a Lepidopteran pest comprising contacting theLepidopteran pest with an inhibitory amount of a chimeric insecticidalprotein of the invention are also contemplated.

In another embodiment, disclosed herein is a transgenic plant cell,plant or plant part comprising a chimeric insecticidal protein, wherein:the chimeric insecticidal protein comprises any amino acid sequence setforth in any of SEQ ID NO: 21, 10, 28, 7, 4, 13, 16, 19, 23, 25, 30, 33,36, 39, 41, 43, 45, 47, 50 or 53; or the chimeric insecticidal proteincomprises a protein having: at least 94% identical to SEQ ID NOs:21, 10;at least 93% identical to SEQ ID NO:28 at least 87% identical to SEQ IDNO:7; at least 90% identity to SEQ ID NO:4; at least 91% identical toSEQ ID NO:13; at least 64% identical to SEQ ID NO:16; at least 66%identical to SEQ ID NO:19; at least 86% identical to SEQ ID NO:23; atleast 91% identical to SEQ ID NO:25; at least 94% identical to SEQ IDNO:30; at least 91% identical to SEQ ID NO:33; at least 64% identical toSEQ ID NO:36; at least 66% identical to SEQ ID NO:39; at least 94%identical to SEQ ID NO:41; at least 84% identical to SEQ ID NO:43; atleast 93% identical to SEQ ID NO:45; at least 94% identical to SEQ IDNO: 47; at least 91% identical to SEQ ID NO:50; or at least 93%identical to SEQ ID NO:53. Methods of controlling a Lepidopteran pestwhich comprise exposing the pest to this transgenic plant cell, plant orplant part, wherein said plant cell, plant or plant part expresses aLepidopteran inhibitory amount of the chimeric insecticidal protein arealso contemplated.

In other embodiments herein, commodity products derived from the plantcell, plant, or plant part wherein the product comprises a detectableamount of the chimeric insecticidal protein are provided. Contemplatedcommodity products include plant biomass, oil, meal, animal feed, flour,flakes, bran, lint, hulls, and processed seed.

Yet another method disclosed herein is a method of producing a seedcomprising a chimeric insecticidal protein, the method comprising:planting at least one seed comprising a chimeric insecticidal protein;growing plants from said seed; and harvesting seed from said plants,wherein said harvested seed comprises the chimeric insecticidal protein.

Recombinant polynucleotide molecules encoding a chimeric insecticidalprotein, comprising a nucleotide sequence selected from the groupconsisting of 1, 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 22, 24,26, 27, 29, 31, 32, 34, 35, 37, 38, 40, 42, 44, 46, 48, 49, 51 or 52;and optionally a polynucleotide sequence encoding an insect inhibitoryagent different from the chimeric insecticidal protein are alsocontemplated herein.

Another recombinant nucleic acid molecule contemplated herein comprisesa heterologous promoter operably linked to a polynucleotide segmentencoding a chimeric insecticidal proteins, wherein: the chimericinsecticidal protein comprises any amino acid sequence set forth in anyof SEQ ID NO: 21, 10, 28, 7, 4, 13, 16, 19, 23, 25, 30, 33, 36, 39, 41,43, 45, 47, 50 or 53; or the chimeric insecticidal protein comprises aprotein having: at least 94% identical to SEQ ID NOs:21, 10; at least93% identical to SEQ ID NO:28; at least 87% identical to SEQ ID NO:7; atleast 90% identity to SEQ ID NO:4; at least 91% identical to SEQ IDNO:13; at least 64% identical to SEQ ID NO:16; at least 66% identical toSEQ ID NO:19; at least 86% identical to SEQ ID NO:23; at least 91%identical to SEQ ID NO:25; at least 94% identical to SEQ ID NO:30; atleast 91% identical to SEQ ID NO:33; at least 64% identical to SEQ IDNO:36; at least 66% identical to SEQ ID NO:39; at least 94% identical toSEQ ID NO:41; at least 84% identical to SEQ ID NO:43; at least 93%identical to SEQ ID NO:45; at least 94% identical to SEQ ID NO: 47; atleast 91% identical to SEQ ID NO:50; or at least 93% identical to SEQ IDNO:53; or the polynucleotide segment hybridizes to a polynucleotidehaving a nucleotide sequence as set forth in any of SEQ ID NO: 1, 2, 3,5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 22, 24, 26, 27, 29, 31, 32, 34,35, 37, 38, 40, 42, 44, 46, 48, 49, 51 or 52.

Other embodiments, features, and advantages of the invention will beapparent from the following detailed description, examples, and claims.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID SEQ ID NO: 1 is a recombinant DNA sequence encoding TIC1100 usedfor expression in a bacterial cell.

SEQ ID NO: 2 is a synthetic DNA sequence encoding TIC1100 for expressionin a plant cell.

SEQ ID NO: 3 is a synthetic DNA sequence encoding TIC1100 for expressionin a plant cell.

SEQ ID NO: 4 is the amino acid sequence of TIC1100.

SEQ ID NO: 5 is a recombinant DNA sequence encoding TIC860 used forexpression in a bacterial cell.

SEQ ID NO: 6 is a synthetic DNA sequence encoding TIC860 for expressionin a plant cell.

SEQ ID NO: 7 is the amino acid sequence of TIC860.

SEQ ID NO: 8 is a recombinant DNA sequence encoding TIC867 used forexpression in a bacterial cell.

SEQ ID NO: 9 is a synthetic DNA sequence encoding TIC867 for expressionin a plant cell.

SEQ ID NO: 10 is the amino acid sequence of TIC867.

SEQ ID NO: 11 is a recombinant DNA sequence encoding TIC867_20 used forexpression in a bacterial cell.

SEQ ID NO: 12 is a synthetic DNA sequence encoding TIC867_20 forexpression in a plant cell.

SEQ ID NO: 13 is the amino acid sequence of TIC867_20.

SEQ ID NO: 14 is a recombinant DNA sequence encoding TIC867_21 used forexpression in a bacterial cell.

SEQ ID NO: 15 is a synthetic DNA sequence encoding TIC867_21 forexpression in a plant cell.

SEQ ID NO: 16 is the amino acid sequence of TIC867_21.

SEQ ID NO: 17 is a recombinant DNA sequence encoding TIC867_22 used forexpression in a bacterial cell.

SEQ ID NO: 18 is a synthetic DNA sequence encoding TIC867_22 forexpression in a plant cell.

SEQ ID NO: 19 is the amino acid sequence of TIC867_22.

SEQ ID NO: 20 is a synthetic DNA sequence encoding TIC867_23 forexpression in the plant cell.

SEQ ID NO: 21 is the amino acid sequence of TIC867_23.

SEQ ID NO: 22 is a synthetic DNA sequence encoding TIC867_24 forexpression in a plant cell.

SEQ ID NO: 23 is the amino acid sequence of TIC867_24.

SEQ ID NO: 24 is a synthetic DNA sequence encoding TIC867_24 forexpression in a plant cell.

SEQ ID NO: 25 is the amino acid sequence of TIC867_25.

SEQ ID NO: 26 is a recombinant DNA sequence encoding TIC868 used forexpression in a bacterial cell.

SEQ ID NO: 27 is a synthetic DNA sequence encoding TIC868 for expressionin a plant cell.

SEQ ID NO: 28 is the amino acid sequence of TIC868.

SEQ ID NO: 29 is a synthetic DNA sequence encoding TIC868_9 forexpression in a plant cell.

SEQ ID NO: 30 is the amino acid sequence of TIC868_9.

SEQ ID NO: 31 is a recombinant DNA sequence encoding TIC868_10 used forexpression in a bacterial cell.

SEQ ID NO: 32 is a synthetic DNA sequence for expression in the plantcell encoding the TIC868 variant, TIC868_10.

SEQ ID NO: 33 is the amino acid sequence of TIC868_10.

SEQ ID NO: 34 is a recombinant DNA sequence encoding TIC868_11 used forexpression in a bacterial cell.

SEQ ID NO: 35 is a synthetic DNA sequence encoding TIC868_11 forexpression in a plant cell.

SEQ ID NO: 36 is the amino acid sequence of TIC868_11.

SEQ ID NO: 37 is a recombinant DNA sequence encoding TIC868_12 used forexpression in a bacterial cell.

SEQ ID NO: 38 is a synthetic DNA sequence encoding TIC868_12 forexpression in the plant cell.

SEQ ID NO: 39 is the amino acid sequence of TIC868_12.

SEQ ID NO: 40 is a synthetic DNA sequence encoding TIC868_13 forexpression in the plant cell.

SEQ ID NO: 41 is the amino acid sequence of TIC868_13.

SEQ ID NO: 42 is a synthetic DNA sequence encoding TIC868_14 forexpression in a plant cell.

SEQ ID NO: 43 is the amino acid sequence of TIC868_14.

SEQ ID NO: 44 is a synthetic DNA sequence encoding TIC868_15 forexpression in a plant cell.

SEQ ID NO: 45 is the amino acid sequence of TIC868_15.

SEQ ID NO: 46 is a synthetic DNA sequence encoding TIC868_29 forexpression in a plant cell.

SEQ ID NO: 47 is the amino acid sequence of TIC868_29.

SEQ ID NO: 48 is a recombinant DNA sequence encoding TIC869 used forexpression in a bacterial cell.

SEQ ID NO: 49 is a synthetic DNA sequence encoding TIC869 for expressionin a plant cell.

SEQ ID NO: 50 is the amino acid sequence of TIC869.

SEQ ID NO: 51 is a recombinant DNA sequence encoding TIC836 used forexpression in a bacterial cell.

SEQ ID NO: 52 is a synthetic DNA sequence encoding TIC836 for expressionin a plant cell.

SEQ ID NO: 53 is the amino acid sequence of TIC836.

DETAILED DESCRIPTION OF THE INVENTION

The problem in the art of agricultural pest control can be characterizedas a need for new insecticidal proteins that are efficacious againsttarget pests, exhibit broad spectrum toxicity against target pestspecies, are capable of being expressed in plants without causingundesirable agronomic issues, and provide an alternative mode of actioncompared to current toxins that are used commercially in plants. Novelchimeric insecticidal proteins are disclosed herein, and address each ofthese needs, particularly against a broad spectrum of Lepidopteraninsect pests.

In order to avoid the development of, or circumvent insect resistanceagainst currently used insecticidal proteins, new insecticidal proteinswith different modes-of-action (MOA), as well as a broad spectrum andefficacy, are needed for Lepidoptera control. One way to address thisneed is to discover new insecticidal proteins from different biologicalsources, preferably from bacteria, fungi or plants. Another approach isto interchange segments between various Bt proteins that exhibitstructural similarities to create new chimeric Bt proteins having insectinhibitory properties. The likelihood of creating a chimeric proteinwith enhanced properties from the re-assortment of the domain structuresof numerous native insecticidal crystal proteins known in the art isknown in the art to be remote. See, e.g. Jacqueline S. Knight, et al. “AStrategy for Shuffling Numerous Bacillus thuringiensis Crystal ProteinDomains.” J. Economic Entomology, 97 (6) (2004): 1805-1813.

Disclosed herein are recombinant nucleic acid molecule sequences thatencode novel chimeric insecticidal proteins. These insecticidal proteinsaddress the ongoing need in the art to engineer additional toxicinsecticidal proteins with improved insecticidal properties such asincreased efficacy against a broader spectrum of target insect pestsspecies and different modes of action. Members of this group ofproteins, including the exemplary proteins disclosed herein, exhibitinsecticidal activity against Lepidopteran insect pest species.

The term “segment” or “fragment” is used in this application to describeconsecutive amino acid or nucleic acid sequences that are shorter thanthe complete amino acid or nucleic acid sequence describing a disclosedchimeric insecticidal protein. A segment or fragment exhibiting insectinhibitory activity is also disclosed in this application if alignmentof such segment or fragment, with the corresponding section of thechimeric insecticidal protein, results in amino acid sequence identityof any fraction percentage from about 65 to about 100 percent betweenthe segment or fragment and the corresponding section of the chimericinsecticidal protein.

Reference in this application to the terms “active” or “activity”,“pesticidal activity” or “pesticidal”, or “insecticidal activity”,“insect inhibitory” or “insecticidal” refer to efficacy of a toxicagent, such as an insecticidal protein, in inhibiting (inhibitinggrowth, feeding, fecundity, or viability), suppressing (suppressinggrowth, feeding, fecundity, or viability), controlling (controlling thepest infestation, controlling the pest feeding activities on aparticular crop containing an effective amount of the insecticidalprotein) or killing (causing the morbidity, mortality, or reducedfecundity of) a pest. These terms are intended to include the result ofproviding a pesticidally effective amount of an insecticidal protein toa pest where the exposure of the pest to the insecticidal proteinresults in morbidity, mortality, reduced fecundity, or stunting. Theseterms also include repulsion of the pest from the plant, a tissue of theplant, a plant part, seed, plant cells, or from the particulargeographic location where the plant may be growing, as a result ofproviding a pesticidally effective amount of the insecticidal protein inor on the plant. In general, pesticidal activity refers to the abilityof an insecticidal protein to be effective in inhibiting the growth,development, viability, feeding behavior, mating behavior, fecundity, orany measurable decrease in the adverse effects caused by an insectfeeding on this protein, protein fragment, protein segment orpolynucleotide of a particular target pest, including but not limited toinsects of the order Lepidoptera. The insecticidal protein can beproduced by the plant or can be applied to the plant or to theenvironment within the location where the plant is located. The terms“bioactivity”, “effective”, “efficacious” or variations thereof are alsoterms interchangeably utilized in this application to describe theeffects of the chimeric insecticidal proteins of the present inventionon target insect pests.

A pesticidally effective amount of a toxic agent, when provided in thediet of a target pest, exhibits pesticidal activity when the toxic agentcontacts the pest. A toxic agent can be an insecticidal protein or oneor more chemical agents known in the art. Insecticidal chemical agentsand insecticidal protein agents can be used alone or in combinationswith each other. Chemical agents include but are not limited to dsRNAmolecules targeting specific genes for suppression in a target pest,organochlorides, organophosphates, carbamates, pyrethroids,neonicotinoids, and ryanoids. Insecticidal protein agents include thechimeric insecticidal proteins set forth in this application, as well asother proteinaceous toxic agents including those that targetLepidopteran pest species, as well as protein toxins that are used tocontrol other plant pests such as Cry proteins available in the art foruse in controlling Coleopteran, Thysanopteranm, Hemipteran andHomopteran species.

It is intended that reference to a pest, particularly a pest of a cropplant, means insect pests of crop plants, particularly thoseLepidopteran insect pests that are controlled by the disclosed chimericinsecticidal proteins. However, reference to a pest can also includeColeopteran, Hemipteran and Homopteran insect pests of plants, as wellas nematodes and fungi when toxic agents targeting these pests areco-localized or present together with the chimeric insecticidal protein,or a protein that is 65 to about 100 percent identical to the chimericinsecticidal protein.

The chimeric insecticidal proteins disclosed herein exhibit insecticidalactivity towards insect pests from the Lepidopteran insect species,including adults, pupae, larvae, and neonates, as well as Hemipteraninsect species, including adults and nymphs. The insects of the orderLepidoptera include, but are not limited to, armyworms, cutworms,loopers, and heliothines in the Family Noctuidae, e.g., fall armyworm(Spodoptera frugiperda), beet armyworm (Spodoptera exigua), berthaarmyworm (Mamestra configurata), black cutworm (Agrotis ipsilon),cabbage looper (Trichoplusia ni), soybean looper (Pseudoplusiaincludens), velvetbean caterpillar (Anticarsia gemmatalis), greencloverworm (Hypena scabra), tobacco budworm (Heliothis virescens),granulate cutworm (Agrotis subterranea), armyworm (Pseudaletiaunipuncta), western cutworm (Agrotis orthogonia); borers, casebearers,webworms, coneworms, cabbageworms and skeletonizers from the FamilyPyralidae, e.g., European corn borer (Ostrinia nubilalis), navelorangeworm (Amyelois transitella), corn root webworm (Crambuscaliginosellus), sod webworm (Herpetogramma licarsisalis), sunflowermoth (Homoeosoma electellum), lesser cornstalk borer (Elasmopalpuslignosellus); leafrollers, budworms, seed worms, and fruit worms in theFamily Tortricidae, e.g., codling moth (Cydia pomonella), grape berrymoth (Endopiza viteana), oriental fruit moth (Grapholita molesta),sunflower bud moth (Suleima helianthana); and many other economicallyimportant Lepidoptera, e.g., diamondback moth (Plutella xylostella),pink bollworm (Pectinophora gossypiella) and gypsy moth (Lymantriadispar). Other insect pests of order Lepidoptera include, e.g., Alabamaargillacea (cotton leaf worm), Archips argyrospila (fruit tree leafroller), Archips rosana (European leafroller) and other Archips species,Chilo suppressalis (Asiatic rice borer, or rice stem borer),Cnaphalocrocis medinalis (rice leaf roller), Crambus caliginosellus(corn root webworm), Crambus teterrellus (bluegrass webworm), Diatraeagrandiosella (southwestern corn borer), Diatraea saccharalis (surgarcaneborer), Earias insulana (spiny bollworm), Earias vittella (spottedbollworm), Helicoverpa armigera (American bollworm), Helicoverpa zea(corn earworm or cotton bollworm), Heliothis virescens (tobaccobudworm), Herpetogramma licarsisalis (sod webworm), Lobesia botrana(European grape vine moth), Phyllocnistis citrella (citrus leafminer),Pieris brassicae (large white butterfly), Pieris rapae (importedcabbageworm, or small white butterfly), Plutella xylostella (diamondbackmoth), Spodoptera exigua (beet armyworm), Spodoptera litura (tobaccocutworm, cluster caterpillar), and Tuta absoluta (tomato leafminer).

Reference in this application to an “isolated DNA molecule”, or anequivalent term or phrase, is intended to mean that the DNA molecule isone that is present alone or in combination with other compositions, butnot within its natural environment. For example, nucleic acid elementssuch as a coding sequence, intron sequence, untranslated leadersequence, promoter sequence, transcriptional termination sequence, andthe like, that are naturally found within the DNA of the genome of anorganism are not considered to be “isolated” so long as the element iswithin the genome of the organism and at the location within the genomein which it is naturally found. However, each of these elements, andsubparts of these elements, would be “isolated” within the scope of thisdisclosure so long as the element is not within the genome of theorganism and at the location within the genome in which it is naturallyfound. Similarly, a nucleotide sequence encoding an insecticidal proteinor any naturally occurring insecticidal variant of that protein would bean isolated nucleotide sequence so long as the nucleotide sequence wasnot within the DNA of the bacterium from which the sequence encoding theprotein is naturally found. A synthetic nucleotide sequence encoding theamino acid sequence of the naturally occurring insecticidal proteinwould be considered to be isolated for the purposes of this disclosure.For the purposes of this disclosure, any transgenic nucleotide sequence,i.e., the nucleotide sequence of the DNA inserted into the genome of thecells of a plant or bacterium, or present in an extrachromosomal vector,would be considered to be an isolated nucleotide sequence whether it ispresent within the plasmid or similar structure used to transform thecells, within the genome of the plant or bacterium, or present indetectable amounts in tissues, progeny, biological samples or commodityproducts derived from the plant or bacterium.

As described further in the Examples, through a chimeragenesis effortabout eight hundred and forty four (844) nucleotide sequences thatencode chimeric insecticidal proteins were constructed from the protoxinand toxin domains of known insecticidal toxins (referred to herein asthe “parent proteins”), and expressed and tested in bioassay forLepidopteran activity. A small number of the constructed chimericinsecticidal proteins exhibited improved Lepidopteran activity or anenhanced Lepidopteran spectrum compared to the parent proteins fromwhich its toxin components were derived.

These novel chimeric insecticidal proteins with improved Lepidopteranactivity or an enhanced Lepidopteran spectrum were constructed from thefollowing insecticidal parent protein protoxin and toxin domains: Cry1Ah(Domain I), Cry1Bb1 (Domains I and II), Cry 1Be2 (Domains I and II),Cry1Ja1 (Domains I and II), Cry1Fa1 (Domains I and II), Cry1Ac (DomainII and protoxin), Cry1Ca (Domain III and protoxin), Cry1Ka (Domain IIIand protoxin), Cry1Jx (Domain III), Cry1Ab (Domain III), Cry1Ab3(protoxin), Cry1Da1(protoxin), Cry4 (protoxin), Cry9 (protoxin), Cry1Be(protoxin), and Cry1Ka (protoxin).

Specifically, the novel chimeric insecticidal proteins of this inventionwith improved Lepidopteran activity or an enhanced Lepidopteran spectrumcomprise the following protoxin and domain combinations: TIC1100/SEQ IDNO:4 (Domain I—Cry1Ah, Domain II—Cry1Ac, Domain III—Cry1Ca,Protoxin—Cry1Ac), TIC860/SEQ ID NO:7 (Domain I—Cry1Bb1, DomainII—Cry1BB1, Domain III—Cry1Ca, Protoxin—Cry1Ac), TIC867/SEQ ID NO:10(Domain I—Cry1Be2, Domain II—Cry1Be2, Domain III—Cry1Ka,Protoxin—Cry1Ab3), TIC868/SEQ ID NO:28 (Domain I—Cry1Be2, DomainII—Cry1Be2, and Domain III—Cry1Ca, Protoxin—Cry1Ab3), TIC869/SEQ IDNO:50 (Domain I—Cry1Ja1, Domain II—Cry1Ja1, Domain III—Cry1Jx,Protoxin—Cry1Ab3) and TIC836/SEQ ID NO:53 (Domain I—Cry1Fa1, DomainII—Cry1Fa1, Domain III—Cry1Ab, Protoxin—Cry1Ac).

Variants in which amino acid substitutions or alternate protoxin domainswere introduced were also constructed for the chimeric insecticidalproteins TIC867 and TIC868. Specifically, these variants of TIC867 andTIC868 comprise the following amino acid substitutions or alternateprotoxin domains: TIC867_20/SEQ ID NO:13 (alternate protoxin domainCry1Da1), TIC867_21/SEQ ID NO:16 (alternate protoxin domain Cry4),TIC867_22/SEQ ID NO:19 (alternate protoxin domain Cry9), TIC867_23/SEQID NO:21 (alternate protoxin domain Cry1Be), TIC867_24/SEQ ID NO:23(alternate protoxin domain Cry1Ka), TIC867_25/SEQ ID NO: 25 (alternateprotoxin domain Cry1Ka), TIC868_9/SEQ ID NO:30 (amino acid modificationN240S_Y343QN349T), TIC868_10/SEQ ID NO:33 (alternate protoxin domainCry1Da1), TIC868_11/SEQ ID NO:36 (alternate protoxin domain Cry4),TIC868_12/SEQ ID NO:39 (alternate protoxin domain Cry9), TIC868_13/SEQID NO:41 (alternate protoxin domain Cry1Be), TIC868_14/SEQ ID NO:43(alternate protoxin domain Cry1Ka), TIC868_15/SEQ ID NO:45 (alternateprotoxin domain Cry1Ca), and TIC868_29/SEQ ID NO:47 (amino acidmodification Q136Y_Y343Q_N349T).

As demonstrated in the Examples, each of these TIC867 and TIC868variants altered the Lepidopteran activity and/or reduced theLepidopteran activity spectrum of the parent chimeric insecticidalprotein, thus indicating that the alternate protoxin domain and theamino acid substitutions had a direct consequence on the insecticidalactivity and spectrum of the chimeric insecticidal proteins TIC867 andTIC868.

Many of the chimeric insecticidal proteins demonstrate insecticidalactivity against multiple Lepidopteran insect pest species.Specifically, the novel chimeric insecticidal proteins disclosed in thisapplication exhibited activity against one or more of the followingLepidopteran insect pests, Velvet bean caterpillar (VBC, Anticarsiagemmatalis), Sugarcane borer (SCB, Diatraea saccharalis), Lessercornstalk borer (LSCB, Elasmopalpus lignosellus), Corn earworm (CEW,Helicoverpa zea), Soybean pod worm (SPW, Helicoverpa zea), Cottonbollworm (CBW, Helicoverpa zea), Tobacco budworm (TBW, Heliothisvirescens), Soybean looper (SBL, Chrysodeixis includens), Black armyworm(BLAW, Spodoptera cosmioides), Southern armyworm (SAW, Spodopteraeridania), Fall armyworm (FAW, Spodoptera frugiperda), Beet armyworm(BAW, Spodoptera exigua), Old World bollworm (OBW, Helicoverpaarmigera), Oriental leafworm (OLW, Spodoptera litura), Pink bollworm(PBW, Pectinophora gossypiella), Southwestern Corn Borer (SWCB, Diatraeagrandiosella), Spotted bollworm (SBW, Earias vitella), American bollworm(SABW, Helicoverpa gelotopeon), and Sunflower looper (SFL, Rachiplusianu). Thus, the exemplary proteins described in this application arerelated by common function and exhibit insecticidal activity towardsinsect pests from the Lepidoptera insect species including adults,larvae and pupae.

Proteins that resemble the chimeric insecticidal proteins can beidentified by comparison to each other using various computer basedalgorithms known in the art. For example, amino acid sequence identitiesof proteins related to the chimeric insecticidal proteins can beanalyzed using a Clustal W alignment using these default parameters:Weight matrix: blosum, Gap opening penalty: 10.0, Gap extension penalty:0.05, Hydrophilic gaps: On, Hydrophilic residues: GPSNDQERK,Residue-specific gap penalties: On (Thompson, et al (1994) Nucleic AcidsResearch, 22:4673-4680). Percent amino acid identity is furthercalculated by the product of 100% multiplied by (amino acididentities/length of the subject protein). Other alignment algorithmsare also available in the art, provide results similar to those obtainedusing Clustal W alignment and are contemplated in this application.

It is intended that a query protein exhibiting insect inhibitoryactivity is disclosed in this application if alignment of such queryprotein with the subject chimeric insecticidal proteins set forth in SEQID NOs: 4, 7, 10, 13, 16, 19, 21, 23, 25, 28, 30, 33, 36, 39, 41, 43,45, 47, 50 and 53 and results in at least about 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or about 100% amino acid sequence identity (or anyfraction percentage in this range) between the query and subjectprotein.

As described further in the Examples of this application, synthetic orartificial sequences encoding the chimeric insecticidal proteins weredesigned for use in plants. Exemplary synthetic nucleotide sequencesthat were designed for use in plants are set forth in SEQ ID NOs: 2 and3 (TIC1100), SEQ ID NO:6 (TIC860), SEQ ID NO:9 (TIC867), SEQ ID NO:12(TIC867_20), SEQ ID NO:15 (TIC867_21), SEQ ID NO:18 (TIC867_22), SEQ IDNO:20 (TIC867_23), SEQ ID NO:22 (TIC867_24), SEQ ID NO: 24 (TIC867_25),SEQ ID NO:27 (TIC868), SEQ ID NO:29 (TIC868_9), SEQ ID NO:32(TIC868_10), SEQ ID NO:35 (TIC868_11), SEQ ID NO:38 (TIC868_12), SEQ IDNO:40 (TIC868_13), SEQ ID NO:42 (TIC868_14), SEQ ID NO:44 (TIC868_15),SEQ ID NO:46 (TIC868_29), SEQ ID NO:49 (TIC869) and SEQ ID NO:52(TIC836).

For expression in plant cells, the chimeric insecticidal proteins can beexpressed to reside in the cytosol or targeted to various organelles ofthe plant cell. For example, targeting a protein to the chloroplast mayresult in increased levels of expressed protein in a transgenic plantwhile preventing off-phenotypes from occurring. Targeting may alsoresult in an increase in pest resistance efficacy in the transgenicevent. A target peptide or transit peptide is a short (3-70 amino acidslong) peptide chain that directs the transport of a protein to aspecific region in the cell, including the nucleus, mitochondria,endoplasmic reticulum (ER), chloroplast, apoplast, peroxisome and plasmamembrane. Some target peptides are cleaved from the protein by signalpeptidases after the proteins are transported. For targeting to thechloroplast, proteins contain transit peptides which are around 40-50amino acids. For descriptions of the use of chloroplast transitpeptides, see U.S. Pat. Nos. 5,188,642 and 5,728,925. Manychloroplast-localized proteins are expressed from nuclear genes asprecursors and are targeted to the chloroplast by a chloroplast transitpeptide (CTP). Examples of such isolated chloroplast proteins include,but are not limited to, those associated with the small subunit (SSU) ofribulose-1,5,-bisphosphate carboxylase, ferredoxin, ferredoxinoxidoreductase, the light-harvesting complex protein I and protein II,thioredoxin F, enolpyruvyl shikimate phosphate synthase (EPSPS), andtransit peptides described in U.S. Pat. No. 7,193,133. It has beendemonstrated in vivo and in vitro that non-chloroplast proteins may betargeted to the chloroplast by use of protein fusions with aheterologous CTP and that the CTP is sufficient to target a protein tothe chloroplast. Incorporation of a suitable chloroplast transit peptidesuch as the Arabidopsis thaliana EPSPS CTP (CTP2) (see, Klee et al.,Mol. Gen. Genet. 210:437-442, 1987) or the Petunia hybrida EPSPS CTP(CTP4) (see, della-Cioppa et al., Proc. Natl. Acad. Sci. USA83:6873-6877, 1986) has been shown to target heterologous EPSPS proteinsequences to chloroplasts in transgenic plants (see, U.S. Pat. Nos.5,627,061; 5,633,435; and 5,312,910; and EP 0218571; EP 189707; EP508909; and EP 924299). For targeting the chimeric insecticidal proteinsto the chloroplast, a sequence encoding a chloroplast transit peptide isplaced 5′ in operable linkage and in frame to a synthetic codingsequence encoding the chimeric insecticidal protein that has beendesigned for optimal expression in plant cells.

Expression cassettes and vectors containing these synthetic orartificial nucleotide sequences were constructed and introduced intocorn, cotton, and soybean plant cells in accordance with transformationmethods and techniques which are known in the art. Transformed cellswere regenerated into transformed plants that were observed to beexpressing the chimeric insecticidal protein. To test pesticidalactivity, bioassays were performed in the presence of Lepidopteran pestlarvae using plant leaf disks obtained from the transformed plants.Recombinant nucleic acid molecule compositions that encode the chimericinsecticidal proteins are contemplated. For example, the chimericinsecticidal proteins can be expressed with recombinant DNA constructsin which a polynucleotide molecule with an ORF encoding a chimericinsecticidal protein is operably linked to genetic expression elementssuch as a promoter and any other regulatory element necessary forexpression in the system for which the construct is intended.Non-limiting examples include a plant-functional promoter operablylinked to the synthetic chimeric insecticidal protein encoding sequencesfor expression of the chimeric insecticidal protein in plants or aBt-functional promoter operably linked to a chimeric insecticidalprotein encoding sequence for expression of the protein in a Btbacterium or other Bacillus species. Other elements can be operablylinked to the chimeric insecticidal proteins encoding sequencesincluding, but not limited to, enhancers, introns, untranslated leaders,encoded protein immobilization tags (HIS-tag), translocation peptides(i.e., plastid transit peptides, signal peptides), polypeptide sequencesfor post-translational modifying enzymes, ribosomal binding sites, andRNAi target sites.

Exemplary recombinant polynucleotide molecules provided herein include,but are not limited to, a heterologous promoter operably linked to apolynucleotide such as SEQ ID NOs: 1, 2, 3, 5, 6, 8, 9, 11, 12, 14, 15,17, 18, 20, 22, 24, 26, 27, 29, 31, 32, 34, 35, 37, 38, 40, 42, 44, 46,48, 49, 51, and 52, that encodes the polypeptide or protein having theamino acid sequence as set forth in SEQ ID NOs: 4 (TIC1100), 7 (TIC860),10 (TIC867), 13 (TIC867_20), 16 (TIC867_21), 19 (TIC867_22), 21(TIC867_23), 23 (TIC867_24), 25 (TIC867_25), 28 (TIC868), 30 (TIC868_9),33 (TIC868_10), 36 (TIC868_11), 39 (TIC867_12), 41 (TIC867_13), 43(TIC867_14), 45 (TIC867_15), 47 (TIC867_29), 50 (TIC869) and 53(TIC836). A heterologous promoter can also be operably linked tosynthetic DNA coding sequences encoding a plastid targeted chimericinsecticidal protein and untargeted chimeric insecticidal protein. It iscontemplated that the codons of a recombinant nucleic acid moleculeencoding for a chimeric insecticidal protein disclosed herein can besubstituted by synonymous codons (known in the art as a silentsubstitution).

A recombinant DNA molecule or construct comprising a chimericinsecticidal protein encoding sequence can further comprise a region ofDNA that encodes for one or more toxic agents which can be configured toconcomitantly express or co-express with a DNA sequence encoding achimeric insecticidal protein, a protein different from a chimericinsecticidal protein, an insect inhibitory dsRNA molecule, or anancillary protein. Ancillary proteins include, but are not limited to,co-factors, enzymes, binding-partners, or other agents that function toaid in the effectiveness of an insect inhibitory agent, for example, byaiding its expression, influencing its stability in plants, optimizingfree energy for oligomerization, augmenting its toxicity, and increasingits spectrum of activity. An ancillary protein may facilitate the uptakeof one or more insect inhibitory agents, for example, or potentiate thetoxic effects of the toxic agent.

A recombinant DNA molecule or construct can be assembled so that allproteins or dsRNA molecules are expressed from one promoter or eachprotein or dsRNA molecule is under separate promoter control or somecombination thereof. The proteins of this invention can be expressedfrom a multi-gene expression system in which a chimeric insecticidalprotein is expressed from a common nucleotide segment which alsocontains other open reading frames and promoters, depending on the typeof expression system selected. For example, a bacterial multi-geneexpression system can utilize a single promoter to drive expression ofmultiply-linked/tandem open reading frames from within a single operon(i.e., polycistronic expression). In another example, a plant multi-geneexpression system can utilize multiply-unlinked expression cassettes,each expressing a different protein or other toxic agent such as one ormore dsRNA molecules.

Recombinant nucleic acid molecules or recombinant DNA constructscomprising chimeric insecticidal protein encoding sequence can bedelivered to host cells by vectors, e.g., a plasmid, baculovirus,synthetic chromosome, virion, cosmid, phagemid, phage, or viral vector.Such vectors can be used to achieve stable or transient expression of achimeric insecticidal protein encoding sequence in a host cell, orsubsequent expression of the encoded polypeptide. An exogenousrecombinant polynucleotide or recombinant DNA construct that compriseschimeric insecticidal protein sequence encoding sequence and that isintroduced into a host cell is referred herein as a “transgene”.

Transgenic bacteria, transgenic plant cells, transgenic plants, andtransgenic plant parts that contain a polynucleotide that encodes anyone or more of the chimeric insecticidal proteins are provided herein.The term “bacterial cell” or “bacterium” can include, but is not limitedto, an Agrobacterium, a Bacillus, an Escherichia, a Salmonella, aPseudomonas, or a Rhizobium cell. The term “plant cell” or “plant” caninclude but is not limited to a dicotyledonous cell or amonocotyledonous cell. Contemplated plants and plant cells include, butare not limited to, alfalfa, banana, barley, bean, broccoli, cabbage,brassica, carrot, cassava, castor, cauliflower, celery, chickpea,Chinese cabbage, citrus, coconut, coffee, corn, clover, cotton, acucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic,grape, hops, leek, lettuce, Loblolly pine, millets, melons, nut, oat,olive, onion, ornamental, palm, pasture grass, pea, peanut, pepper,pigeonpea, pine, potato, poplar, pumpkin, Radiata pine, radish,rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum, Southernpine, soybean, spinach, squash, strawberry, sugar beet, sugarcane,sunflower, sweet corn, sweet gum, sweet potato, switchgrass, tea,tobacco, tomato, triticale, turf grass, watermelon, and wheat plant cellor plant. In certain embodiments, transgenic plants and transgenic plantparts regenerated from a transgenic plant cell are provided. In certainembodiments, the transgenic plants can be obtained from a transgenicseed, by cutting, snapping, grinding or otherwise disassociating thepart from the plant. In certain embodiments, the plant part can be aseed, a boll, a leaf, a flower, a stem, a root, or any portion thereof,or a non-regenerable portion of a transgenic plant part. As used in thiscontext, a “non-regenerable” portion of a transgenic plant part is aportion that cannot be induced to form a whole plant or that cannot beinduced to form a whole plant that is capable of sexual and/or asexualreproduction. In certain embodiments, a non-regenerable portion of aplant part is a portion of a transgenic seed, boll, leaf, flower, stem,or root.

Methods of making transgenic plants that comprise Lepidoptera-inhibitoryamounts of a chimeric insecticidal proteins are provided. Such plantscan be made by introducing a polynucleotide that encodes the chimericinsecticidal proteins provided in this application into a plant cell,and selecting a plant derived from said plant cell that expresses aninsect or Lepidoptera-inhibitory amount of the chimeric insecticidalprotein. Plants can be derived from the plant cells by regeneration,seed, pollen, or meristem transformation techniques. Methods fortransforming plants are known in the art. For example,Agrobacterium-mediated transformation is described in U.S. PatentApplication Publications 2009/0138985A1 (soybean), 2008/0280361A1(soybean), 2009/0142837A1 (corn), 2008/0282432 (cotton), and2008/0256667 (cotton).

Plants expressing the chimeric insecticidal proteins can be crossed bybreeding with transgenic events expressing other insecticidal proteinsand/or expressing other transgenic traits such as other insect controltraits, herbicide tolerance genes, genes conferring yield or stresstolerance traits, and the like, or such traits can be combined in asingle vector so that the traits are all linked.

Processed plant products, wherein the processed product comprises adetectable amount of a chimeric insecticidal protein, an insectinhibitory segment or fragment thereof, or any distinguishing portionthereof, are also disclosed in this application. In certain embodiments,the processed product is selected from the group consisting of plantparts, plant biomass, oil, meal, sugar, animal feed, flour, flakes,bran, lint, hulls, processed seed, and seed. In certain embodiments, theprocessed product is non-regenerable. The plant product can comprisecommodity or other products of commerce derived from a transgenic plantor transgenic plant part, where the commodity or other products can betracked through commerce by detecting nucleotide segments or expressedRNA or proteins that encode or comprise distinguishing portions of achimeric insecticidal protein.

Methods of controlling insects, in particular Lepidoptera infestationsof crop plants, with the chimeric insecticidal proteins are alsodisclosed in this application. Such methods can comprise growing a plantcomprising an insect- or Lepidoptera-inhibitory amount of the chimericinsecticidal protein. In certain embodiments, such methods can furthercomprise any one or more of: (i) applying any composition comprising orencoding a chimeric insecticidal protein to a plant or a seed that givesrise to a plant; and (ii) transforming a plant or a plant cell thatgives rise to a plant with a polynucleotide encoding a chimericinsecticidal protein. In general, it is contemplated that chimericinsecticidal protein can be provided in a composition, provided in amicroorganism, or provided in a transgenic plant to confer insectinhibitory activity against Lepidopteran insects.

In certain embodiments, the chimeric insecticidal protein is theinsecticidally active ingredient of an insect inhibitory compositionprepared by culturing recombinant Bacillus or any other recombinantbacterial cell transformed to express a chimeric insecticidal proteinunder conditions suitable for expression. Such a composition can beprepared by desiccation, lyophilization, homogenization, extraction,filtration, centrifugation, sedimentation, or concentration of a cultureof such recombinant cells expressing/producing the chimeric insecticidalprotein. Such a process can result in a Bacillus or otherentomopathogenic bacterial cell extract, cell suspension, cellhomogenate, cell lysate, cell supernatant, cell filtrate, or cellpellet. By obtaining the chimeric insecticidal protein so produced, acomposition that includes the chimeric insecticidal protein can includebacterial cells, bacterial spores, and parasporal inclusion bodies andcan be formulated for various uses, including as agricultural insectinhibitory spray products or as insect inhibitory formulations in dietbioassays.

The aforementioned compound or formulation can further comprise anagriculturally-acceptable carrier, such as a bait, a powder, dust,pellet, granule, spray, emulsion, a colloidal suspension, an aqueoussolution, a Bacillus spore or crystal preparation or a seed treatment.The compound or formulation can also further comprise a recombinantplant cell, plant tissue, seed or plant transformed to express one ormore of the proteins; or bacterium transformed to express one or more ofthe proteins. Depending on the level of insect inhibitory orinsecticidal inhibition inherent in the recombinant polypeptide and thelevel of compound or formulation to be applied to a plant or diet assay,the compound or formulation can include various by weight amounts of therecombinant polypeptide, e.g. from 0.0001% to 0.001% to 0.01% to 1% to99% by weight of the recombinant polypeptide.

In an embodiment, in order to reduce the likelihood of resistancedevelopment, an insect inhibitory composition or transgenic plantcomprising a chimeric insecticidal protein can further comprise at leastone additional toxic agent that exhibits insect inhibitory activityagainst the same Lepidopteran insect species, but which is differentfrom the chimeric insecticidal protein. Possible additional toxic agentsfor such a composition include an insect inhibitory protein and aninsect inhibitory dsRNA molecule. One example for the use of suchribonucleotide sequences to control insect pests is described in Baum,et al. (U.S. Patent Publication 2006/0021087 A1). Such additionalpolypeptide(s) for the control of Lepidopteran pests may be selectedfrom the group consisting of an insect inhibitory protein, such as, butnot limited to, Cry1A (U.S. Pat. No. 5,880,275), Cry1Ab, Cry1Ac,Cry1A.105, Cry1Ae, Cry1B (U.S. patent Publication Ser. No. 10/525,318),Cry1C (U.S. Pat. No. 6,033,874), Cry1D, Cry1E, Cry1F, and Cry1A/Fchimeras (U.S. Pat. Nos. 7,070,982; 6,962,705; and 6,713,063), Cry1G,Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry2A, Cry2Ab (U.S. Pat. No.7,064,249), Cry2Ae, Cry4B, Cry6, Cry7, Cry8, Cry9, Cry15, Cry43A,Cry43B, Cry51Aa1, ET66, TIC400, TIC800, TIC834, TIC1415, Vip3A, VIP3Ab,VIP3B, AXMI-001, AXMI-002, AXMI-030, AXMI-035, AND AXMI-045 (U.S. PatentPublication 2013-0117884 A1), AXMI-52, AXMI-58, AXMI-88, AXMI-97,AXMI-102, AXMI-112, AXMI-117, AXMI-100 (U.S. Patent Publication2013-0310543 A1), AXMI-115, AXMI-113, AXMI-005 (U.S. Patent Publication2013-0104259 A1), AXMI-134 (U.S. Patent Publication 2013-0167264 A1),AXMI-150 (U.S. Patent Publication 2010-0160231 A1), AXMI-184 (U.S.Patent Publication 2010-0004176 A1), AXMI-196, AXMI-204, AXMI-207,AXMI-209 (U.S. Patent Publication 2011-0030096 A1), AXMI-218, AXMI-220(U.S. Patent Publication 2014-0245491 A1), AXMI-221z, AXMI-222z,AXMI-223z, AXMI-224z, AXMI-225z (U.S. Patent Publication 2014-0196175A1), AXMI-238 (U.S. Patent Publication 2014-0033363 A1), AXMI-270 (U.S.Patent Publication 2014-0223598 A1), AXMI-345 (U.S. Patent Publication2014-0373195 A1), DIG-3 (U.S. Patent Publication 2013-0219570 A1), DIG-5(U.S. Patent Publication 2010-0317569 A1), DIG-11 (U.S. PatentPublication 2010-0319093 A1), AfIP-1A and derivatives thereof (U.S.Patent Publication 2014-0033361 A1), AfIP-1B and derivatives thereof(U.S. Patent Publication 2014-0033361 A1), PIP-1APIP-1B (U.S. PatentPublication 2014-0007292 A1), PSEEN3174 (U.S. Patent Publication2014-0007292 A1), AECFG-592740 (U.S. Patent Publication 2014-0007292A1), Pput_1063 (U.S. Patent Publication 2014-0007292 A1), Pput_1064(U.S. Patent Publication 2014-0007292 A1), GS-135 and derivativesthereof (U.S. Patent Publication 2012-0233726 A1), GS153 and derivativesthereof (U.S. Patent Publication 2012-0192310 A1), GS154 and derivativesthereof (U.S. Patent Publication 2012-0192310 A1), GS155 and derivativesthereof (U.S. Patent Publication 2012-0192310 A1), SEQ ID NO:2 andderivatives thereof as described in U.S. Patent Publication 2012-0167259A1, SEQ ID NO:2 and derivatives thereof as described in U.S. PatentPublication 2012-0047606 A1, SEQ ID NO:2 and derivatives thereof asdescribed in U.S. Patent Publication 2011-0154536 A1, SEQ ID NO:2 andderivatives thereof as described in U.S. Patent Publication 2011-0112013A1, SEQ ID NO:2 and 4 and derivatives thereof as described in U.S.Patent Publication 2010-0192256 A1, SEQ ID NO:2 and derivatives thereofas described in U.S. Patent Publication 2010-0077507 A1, SEQ ID NO:2 andderivatives thereof as described in U.S. Patent Publication 2010-0077508A1, SEQ ID NO:2 and derivatives thereof as described in U.S. PatentPublication 2009-0313721 A1, SEQ ID NO:2 or 4 and derivatives thereof asdescribed in U.S. Patent Publication 2010-0269221 A1, SEQ ID NO:2 andderivatives thereof as described in U.S. Pat. No. 7,772,465 (B2),CF161_0085 and derivatives thereof as described in WO2014/008054 A2,Lepidopteran toxic proteins and their derivatives as described in USPatent Publications US2008-0172762 A1, US2011-0055968 A1, andUS2012-0117690 A1; SEQ ID NO:2 and derivatives thereof as described inU.S. Pat. No. 7,510,878(B2), SEQ ID NO:2 and derivatives thereof asdescribed in U.S. Pat. No. 7,812,129(B1); and the like.

In other embodiments, an insect inhibitory composition or transgenicplant can further comprise at least one additional toxic agent thatexhibits insect inhibitory activity to an insect pest that is notinhibited by the chimeric insecticidal proteins of the present invention(such as Coleopteran, Hemipteran and Homopteran pests), in order toexpand the spectrum of insect inhibition obtained.

Such additional toxic agent for the control of Coleopteran pests may beselected from the group consisting of an insect inhibitory protein, suchas, but not limited to, Cry3Bb (U.S. Pat. No. 6,501,009), Cry1Cvariants, Cry3A variants, Cry3, Cry3B, Cry34/35, 5307, AXMI134 (U.S.Patent Publication 2013-0167264 A1) AXMI-184 (U.S. Patent Publication2010-0004176 A1), AXMI-205 (U.S. Patent Publication 2014-0298538 A1),axmi207 (U.S. Patent Publication 2013-0303440 A1), AXMI-218, AXMI-220(U.S. Patent Publication 20140245491A1), AXMI-221z, AXMI-223z (U.S.Patent Publication 2014-0196175 A1), AXMI-279 (U.S. Patent Publication2014-0223599 A1), AXMI-R1 and variants thereof (U.S. Patent Publication2010-0197592 A1, TIC407, TIC417, TIC431, TIC807, TIC853, TIC901,TIC1201, TIC3131, DIG-10 (U.S. Patent Publication 2010-0319092 A1),eHIPs (U.S. Patent Application Publication No. 2010/0017914), IP3 andvariants thereof (U.S. Patent Publication 2012-0210462 A1), andω-Hexatoxin-Hv1a (U.S. Patent Application Publication US2014-0366227A1).

Such additional toxic agent for the control of Hemipteran pests may beselected from the group consisting of Hemipteran-active proteins suchas, but not limited to, TIC1415 (US Patent Publication 2013-0097735 A1),TIC807 (U.S. Pat. No. 8,609,936), TIC834 (U.S. Patent Publication2013-0269060 A1), AXMI-036 (U.S. Patent Publication 2010-0137216 A1),and AXMI-171 (U.S. Patent Publication 2013-0055469 A1). Additionalpolypeptides for the control of Coleopteran, Lepidopteran, andHemipteran insect pests can be found on the Bacillus thuringiensis toxinnomenclature website maintained by Neil Crickmore (on the world wide webat btnomenclature.info).

Chimeric insecticidal protein-encoding sequences and sequences having asubstantial percentage identity to the chimeric insecticidal proteinscan be identified using methods known to those of ordinary skill in theart such as polymerase chain reaction (PCR), thermal amplification andhybridization. For example, the chimeric insecticidal proteins can beused to produce antibodies that bind specifically to related proteins,and can be used to screen for and to find other proteins that areclosely related.

Furthermore, nucleotide sequences encoding the chimeric insecticidalproteins can be used as probes and primers for screening to identifyother members of the class using thermal-cycle or isothermalamplification and hybridization methods. For example, oligonucleotidesderived from sequences as set forth in SEQ ID NO:2 can be used todetermine the presence or absence of a chimeric insecticidal transgenein a deoxyribonucleic acid sample derived from a commodity product.Given the sensitivity of certain nucleic acid detection methods thatemploy oligonucleotides, it is anticipated that oligonucleotides derivedfrom sequences as set forth in any of SEQ ID NO:2 can be used to detectthe respective chimeric insecticidal protein in commodity productsderived from pooled sources where only a fraction of the commodityproduct is derived from a transgenic plant containing any of SEQ IDNO:2.

EXAMPLES

In view of the foregoing, those of skill in the art will appreciate thatthe following disclosed embodiments are merely representative of theinvention, which may be embodied in various forms. Thus, specificstructural and functional details disclosed herein are not to beinterpreted as limiting.

Example 1 Creation and Cloning of Lepidopteran-Active Novel ChimericInsecticidal Protein Coding Sequences

This Example illustrates the creation of the novel chimeric insecticidalproteins and the cloning and expressing of the chimeric insecticidalproteins.

Recombinant nucleic acid sequences were constructed from known Cryprotein genes to produce polynucleotide sequences encoding novelchimeric insecticidal proteins. The resulting polynucleotide sequenceswere cloned into a Bacillus thuringiensis (Bt) expression plasmidvector. After confirmation of the polynucleotide sequence, theexpression plasmid was transformed into Bt and expressed. Preparationsof the expressed novel chimeric proteins were assayed for activityagainst various Lepidopteran pests.

Many polynucleotide sequences encoding chimeric insecticidal proteinswere produced and tested in bioassay. Not all of the chimericinsecticidal proteins demonstrated activity. Only a few of the chimericinsecticidal proteins were selected based upon their activity tospecific Lepidoptera demonstrated in bioassay. Amino acid variants inwhich amino acid substitutions, or alternate protoxin domains, wereintroduced were also produced based upon the original chimericinsecticidal proteins TIC867 and TIC868. The components of the chimericinsecticidal proteins (domains I, II and III and the protoxin) of thepresent invention are presented in Table 1. The amino acid substitutionsin the TIC868 variants relative to the original TIC868 protein sequenceare also presented.

TABLE 1 Novel chimeric pesticidal proteins and their components. PRT SEQID Amino Acid Toxin NO: Dom1 Dom2 Dom3 Protox Modifications* TIC1100 4Cry1Ah Cry1Ac Cry1Ca Cry1Ac TIC860 7 Cry1Bb1 Cry1Bb1 Cry1Ca Cry1AcTIC867 10 Cry1Be2 Cry1Be2 Cry1Ka Cry1Ab3 TIC867_20 13 Cry1Be2 Cry1Be2Cry1Ka Cry1Da1 TIC867_21 16 Cry1Be2 Cry1Be2 Cry1Ka Cry4 TIC867_22 19Cry1Be2 Cry1Be2 Cry1Ka Cry9 TIC867_23 21 Cry1Be2 Cry1Be2 Cry1Ka Cry1BeTIC867_24 23 Cry1Be2 Cry1Be2 Cry1Ka Cry1Ka TIC867_25 25 Cry1Be2 Cry1Be2Cry1Ka Cry1Ca TIC868 28 Cry1Be2 Cry1Be2 Cry1Ca Cry1Ab3 TIC868_9 30Cry1Be2 Cry1Be2 Cry1Ca Cry1Ab3 N240S_Y343Q_N349T TIC868_10 33 Cry1Be2Cry1Be2 Cry1Ca Cry1Da1 TIC868_11 36 Cry1Be2 Cry1Be2 Cry1Ca Cry4TIC868_12 39 Cry1Be2 Cry1Be2 Cry1Ca Cry9 TIC868_13 41 Cry1Be2 Cry1Be2Cry1Ca Cry1Be TIC868_14 43 Cry1Be2 Cry1Be2 Cry1Ca Cry1Ka TIC868_15 45Cry1Be2 Cry1Be2 Cry1Ca Cry1Ca TIC868_29 47 Cry1Be2 Cry1Be2 Cry1CaCry1Ab3 Q136Y_Y343Q_N349T TIC869 50 Cry1Ja1 Cry1Ja1 Cry1Jx Cry1Ab3TIC836 53 Cry1Fa1 Cry1Fa1 Cry1Ab Cry1Ac *The amino acid mutations areidentified using the standard IUPAC amino acid code. See IUPAC-IUB JointCommission on Biochemical Nomenclature. Nomenclature and Symbolism forAmino Acids and Peptides. Eur. J. Biochem. 138: 9-37 (1984). The firstamino acid sequence abbreviation indicates the original amino acid inthe given scaffold protein, the number represents the position of theamino acid, and the second amino acid sequence abbreviation indicatesthe amino acid placed in that position in the improved variant protein.

Example 2 The Novel Chimeric Insecticidal Proteins Demonstrate ActivityAgainst Lepidopteran Pests

This Example illustrates the testing of the chimeric insecticidalproteins described in Example 1 and the Lepidopteran activity observedfor the chimeric insecticidal proteins.

Polynucleotide sequences encoding chimeric insecticidal proteins wereexpressed in Bt. The expressed chimeric insecticidal proteins were thenassayed against a variety of Lepidoptera known to be pests of corn,sugarcane, soybean and cotton, as well as other crop plants.Specifically, the insecticidal proteins were assayed for activityagainst Velvetbean caterpillar (VBC, Anticarsia gemmatalis), Sugarcaneborer (SCB, Diatraea saccharalis), Lesser cornstalk borer (LSCB,Elasmopalpus lignosellus), Corn earworm (CEW, Helicoverpa zea), Tobaccobudworm (TBW, Heliothis virescens), Soybean looper (SBL, Chrysodeixisincludens), Black armyworm (BLAW, Spodoptera cosmioides), Southernarmyworm (SAW, Spodoptera eridania), Fall armyworm (FAW, Spodopterafrugiperda), Beet armyworm (BAW, Spodoptera exigua), Old World bollworm(OBW, Helicoverpa armigera), Oriental leafworm (OLW, Spodoptera litura),Pink bollworm (PBW, Pectinophora gossypiella), Black cutworm (BCW,Agrotis ipsilon), Southwestern Corn Borer (SWCB, Diatraea grandiosella),Spotted bollworm (SBW, Earias vitella), and European corn borer (ECB,Ostrinia nubilalis). Corn earworm (CEW, Helicoverpa zea) is alsoreferred to as Soybean pod worm (SPW) and Cotton bollworm (CBW).Activity was determined through a combination of mortality and stuntingscores as well as MIC50 scores. MIC50 refers to a molt inhibitionconcentration wherein both the dead larvae and L1 larvae (larvae thatfailed to molt to second instars) are factored into the score. Table 2shows the activity of each chimeric insecticidal protein. A ‘+’ signindicates activity observed to the specific insect pest.

TABLE 2 Bioassay activity against selected Lepidoptera. PRT Insect SEQCEW ID SPW Toxin NO: VBC SCB LSCB CBW BLAW TBW SBL SAW FAW BAW OBW OLWPBW BCW SWCB ECB SBW TIC1100 4 + + + + + + + TIC8607 + + + + + + + + + + + + + TIC867 10 + + + + + + + + + TIC867_20 13TIC867_21 16 + TIC867_22 19 + + TIC868 28 + + + + + + + + + + TIC868_1033 + TIC868_11 36 + TIC868_12 39 + TIC869 50 + + + + + TIC836 53 + + + ++

As can be seen in Table 2 above, most of the chimeric insecticidalproteins exhibited activity against one or more Lepidopteran pestspecies.

Example 3 Synthesis of Genes Encoding Chimeric Insecticidal Proteins andfor Expression in Plants

This Example illustrates the synthesis of polynucleotides encoding thechimeric insecticidal proteins for expression in plants.

Synthetic coding sequences were constructed for use in expression of thechimeric insecticidal proteins in plants. The synthetic sequences weredesigned and synthesized according to methods generally described inU.S. Pat. No. 5,500,365, avoiding certain inimical problem sequencessuch as ATTTA and A/T rich plant polyadenylation sequences whilepreserving the amino acid sequence of the chimeric insecticidal protein.The nucleotide sequences for these genes encoding the chimericinsecticidal proteins for expression in plants are listed in Table 3.

TABLE 3 Polynucleotide Sequences Encoding Chimeric Insecticidal ProteinsDesigned for Use in Plants. DNA PRT Insecticidal SEQ ID SEQ ID ProteinNO: NO: TIC1100 2 4 TIC1100 3 4 TIC860 6 7 TIC867 9 10 TIC867_20 12 13TIC867_21 15 16 TIC867_22 18 19 TIC867_23 20 21 TIC867_24 22 23TIC867_25 24 25 TIC868 27 28 TIC868_9 29 30 TIC868_10 32 33 TIC868_11 3536 TIC868_12 38 39 TIC868_13 40 41 TIC868_14 42 43 TIC868_15 44 45TIC868_29 46 47 TIC869 49 50 TIC836 52 53

Example 4 Expression Cassettes for the Expression of ChimericInsecticidal Proteins in Plants

This Example illustrates the construction of expression cassettescomprising polynucleotide sequences designed for use in plants whichencode chimeric insecticidal proteins.

A variety of plant expression cassettes were constructed with thepolynucleotide sequences encoding the chimeric insecticidal proteinsdesigned for plant expression provided in Table 3. Such expressioncassettes are useful for transient expression in plant protoplasts ortransformation of plant cells. Typical expression cassettes weredesigned with respect to the eventual placement of the protein withinthe cell. One set of expression cassettes was designed in a manner toallow the protein to be translated and remain in the cytosol. Anotherset of expression cassettes was designed to have a transit peptidecontiguous with the toxin protein to allow targeting to an organelle ofthe cell such as the chloroplast or plastid. All expression cassetteswere designed to begin at the 5′ end with a promoter, which can becomprised of multiple promoter elements, enhancer elements, or otherexpression elements known to those of ordinary skill in the art operablylinked to boost the expression of the transgene. The promoter sequencewas usually followed contiguously with one or more leader sequences 3′to the promoter. An intron sequence was usually provided 3′ to theleader sequence to improve expression of the transgene. A codingsequence for the toxin or transit peptide and coding sequence for thetoxin was usually located 3′ to the operably linked promoter, leader andintron configuration. A 3′UTR sequence was usually provided 3′ of thecoding sequence to facilitate termination of transcription and toprovide sequences important for the polyadenylation of the resultingtranscript. All of the elements described above were operably linked andarranged sequentially, often with additional sequences provided for theconstruction of the expression cassette.

Example 5 Lepidopteran Activity of the Chimeric Insecticidal Proteins inStably Transformed Corn

This Example illustrates the inhibitory activity exhibited by thechimeric insecticidal proteins against Lepidopteran pests when expressedin corn plants and provided as a diet to the respective corn insectpest.

Corn variety LH244 was transformed with the binary transformationvectors described in Example 4 using an Agrobacterium-mediatedtransformation method. The transformed cells were induced to form plantsby methods known in the art. Bioassays using plant leaf disks wereperformed analogous to those described in U.S. Pat. No. 8,344,207. Anon-transformed LH244 plant was used to obtain tissue to be used as anegative control. Multiple transformation events from each binary vectorwere assessed against Corn earworm (CEW, Helicoverpa zea), Fall armyworm(FAW, Spodoptera frugiperda), Black cutworm (BCW, Agrotis ipsilon) andSouthwestern Corn Borer (SWCB, Diatraea grandiosella).

Leaf disc bioassay was performed on R₀ and F₁ generation transgenicplants. In addition, leaf damage ratings were assessed for wholetransgenic F₁ plants expressing certain chimeric insecticidal proteinsinfested with the Lepidopteran insect pests. F₁ transgenic eventsexpressing TIC860 and TIC868 were also assessed for activity in thefield against FAW, CEW, and SWCB. The assay results are shown in Table4. A ‘+’ sign indicates activity observed to the specific insect pest.As can be seen in Table 4, most of the chimeric insecticidal proteinsand many of the chimeric insecticidal protein variants demonstratedactivity against one or more Lepidopteran pest species.

TABLE 4 Bioassay activity of chimeric insecticidal proteins from stablytransformed corn leaf tissue. PRT Insect SEQ CEW ID SPW Toxin NO: VBCSCB LSCB CBW BLAW TBW SBL SAW FAW BAW OBW OLW PBW BCW SWCB ECB SBWTIC1100 4 + + + + + + + TIC860 7 + + + + + + + + + + + + + TIC86710 + + + + + + + + + TIC867_20 13 NT NT NT NT NT NT NT NT NT NT NT NT NTNT NT TIC867_21 16 NT NT NT + NT NT NT NT NT NT NT NT NT NT NT NTTIC867_22 19 NT NT NT + NT NT NT NT + NT NT NT NT NT NT NT NT TIC86828 + + + + + + + + + + + TIC868_10 33 NT NT NT + NT NT NT NT + NT NT NTNT NT NT NT NT TIC868_11 36 NT NT NT + NT NT NT NT + NT NT NT NT NT NTNT NT TIC868_12 39 NT NT NT + NT NT NT NT + NT NT NT NT NT NT NT NTTIC869 50 + + + + + + TIC836 53 + + + + +

Example 6 Lepidopteran Activity of the Chimeric Insecticidal Proteins inStably Transformed Soybean

This Example illustrates the inhibitory activity exhibited by thechimeric insecticidal proteins against Lepidopteran pests when expressedin soybean plants and provided as a diet to the respective insect pest.

The coding sequences for selected chimeric insecticidal proteins wereredesigned for plant expression, cloned into a binary planttransformation vector, and used to transform soybean plant cells. Theplant transformation vectors comprised a first transgene cassette forexpression of the chimeric insecticidal protein as described in Example4 and a second transgene cassette for the selection of transformed plantcells using spectinomycin selection. In some instances, such as in thecase of TIC1100, TIC860 and TIC836, a chloroplast transit peptide codingsequence was operably linked to the chimeric insecticidal codingsequence. Assays were performed with plastid targeted and untargetedTIC1100, TIC860 and TIC836. Table 5 below shows the chimericinsecticidal and TIC867 variant chimeric insecticidal protein andassociated coding sequences used for expression in stably transformedsoybean.

Soybean plant cells were transformed using the binary transformationvectors described above by Agrobacterium-mediated transformation. Theresulting transformed plant cells were induced to form whole soybeanplants. Leaf tissue was harvested and used in bioassay as described inExample 5 or alternatively, lyophilized tissue was used in the insectdiet for bioassay. Bioassay was performed against FAW, Southern armyworm(SAW, Spodoptera eridania), Soybean looper (SBL, Chrysodeixisincludens), Soybean Pod Worm (SPW, Helicoverpa zea), Velvetbeancaterpillar (VBC, Anticarsia gemmatalis), Tobacco budworm (TBW,Heliothis virescens), Black armyworm (BLAW, Spodoptera cosmioides),Lesser cornstalk borer (LSCB, Elasmopalpus lignosellus) and Old Worldbollworm (OBW, Helicoverpa armigera).

Table 5 shows the activity against selected species of Lepidoptera foreach insecticidal protein in R₀ generation plants, wherein ‘+’ indicatesactivity. As can be seen in Table 5, each of the chimeric insecticidalproteins expressed in stably transformed soybean demonstrated activityagainst multiple Lepidopteran species. Of particular note is that theTIC867 variant, TIC867_23 demonstrated activity against SPW.

TABLE 5 Bioassay activity of chimeric insecticidal proteins from stablytransformed R₀ soybean leaf tissue. Insecticidal Protein FAW SAW SBL SPWVBC TBW BLAW LSCB OBW TIC1100 + + + + + + + TIC860 + + + + +TIC867 + + + + + + TIC867_20 + + TIC867_21 + + TIC867_22 + +TIC867_23 + + + + TIC867_24 + + TIC867_25 + + TIC868 + + + + +TIC869 + + + + TIC836 + + + + + + +

Selected transformed events were allowed to self-pollinate and theresulting seed was grown. Leaf tissue was harvested from the R₁generation plants and used in a feeding bioassay. R₁ plants expressingTIC1100, TIC860, TIC867, TIC868, TIC869 and TIC836 were assayed foractivity against SAW, SBL, SPW and VBC. Table 6 shows the activityobserved in these tests. A ‘+’ sign indicates activity observed to thespecific insect pest. As demonstrated in Table 6, most of the expressedchimeric insecticidal proteins from R₁ generation plants demonstratedactivity to one or more Lepidopteran species.

TABLE 6 Bioassay activity of chimeric insecticidal proteins from stablytransformed R₁ soybean leaf tissue. Toxin SAW SBL SPW VBC TIC1100 + + +TIC860 + + + TIC867 + TIC868 + + + TIC869 + + + TIC836 + + +

Table 7 demonstrates the results of field tests conducted in screenhouses with stably transformed R₁ generation soybean plants expressingTIC1100, TIC860, and TIC836. Species used to infest plants in the screenhouses include SAW, SBL and SPW. Resistance was defined as being lessthan or equal to fifteen percent defoliation in the soybean plants. Theresistance observed in these cage trials is consistent with theresistance observed in the R₁ generation soybean leaf tissue assaypresented in Table 6. A ‘+’ sign indicates activity observed to thespecific insect pest.

TABLE 7 Activity Profile of TIC1100, TIC860 and TIC836 Expressed in R₁Generation Soybean Tested in Screen House Field Tests. Toxin SAW SBL SPWTIC1100 + + TIC860 + + TIC836 + +

Field tests in screen houses with stably transformed R₁ generationsoybean plants expressing TIC867 and TIC869 were also conducted at twodifferent locations in Argentina, Acevedo and Fontezuela. Species usedto infest plants in the screen houses include South American bollworm(SABW, Helicoverpa gelotopeon), VBC, BLAW, and Sunflower looper (SFL,Rachiplusia nu). Resistance was defined as being less than or equal tofifteen percent defoliation in the soybean plants. Table 8 below showsthe resistance observed. A ‘+’ sign indicates activity observed to thespecific insect pest. As demonstrated in Table 8, transgenic soybeanplants expressing TIC867 demonstrated resistance to BLAW and VBC.Transgenic soybean plants expressing TIC869 demonstrated resistance toSABW, SFL, BLAW, and VBC.

TABLE 8 Activity Profile of TIC867 and TIC869 Expressed in R₁ GenerationSoybean Tested in Screen House Field Tests. Acevedo Fontezuela ToxinSABW SFL VBC SABW BLAW VBC TIC867 + + + TIC869 + + + + +

Example 7 Lepidopteran Activity of the Chimeric Insecticidal Proteins inStably Transformed Cotton

This Example illustrates the inhibitory activity exhibited by thechimeric insecticidal proteins against Lepidopteran pests when expressedin cotton plants and provided as a diet to the respective insect pest.

The coding sequences for selected chimeric insecticidal proteins wereredesigned for plant expression, cloned into a binary planttransformation vector, and used to transform cotton plant cells. Theresulting binary vectors were similar to those described in Example 4and were used to express plastid targeted and untargeted TIC860 (codingsequence: SEQ ID NO: 6; protein sequence: SEQ ID NO: 7), TIC867 (codingsequence: SEQ ID NO: 9; protein sequence: SEQ ID NO: 10), TIC868 (codingsequence: SEQ ID NO: 27; protein sequence: SEQ ID NO: 28) and TIC867_23(coding sequence: SEQ ID NO: 20; protein sequence: SEQ ID NO: 23).

Cotton plant cells were transformed by an Agrobacterium-mediatedtransformation method. Transformed cotton cells were induced to formwhole plants. Cotton leaf tissue was used in bioassay as described inExample 5 against Cotton Boll Worm (CBW, Helicoverpa zea), FAW, TBW andSBL. Table 9 shows the activity observed against these Lepidopteranspecies for TIC860, TIC867, and TIC868 in stably transformed R₀generation cotton, wherein ‘+’ indicate activity. As can be seen inTable 9, TIC860, TIC867, and TIC868 demonstrated activity against two ormore Lepidopteran pest species in stably transformed R₀ generationcotton.

TABLE 9 Bioassay activity of TIC860, TIC867 and TIC868 from stablytransformed R₀ cotton leaf tissue. Toxin CBW FAW TBW SBL TIC860 + +TIC867 + + + NT TIC868 + +

Selected transformation events were used to produce R₁ seed. R₁ Plantsexpressing TIC860, TIC867, and TIC868 were assayed for resistance toCBW, FAW, TBW, and SBL. Leaf, square and boll tissues were used inassay. Table 10 shows the activity observed in these tests. A ‘+’ signindicates activity observed to the specific insect pest. As demonstratedin Table 10, TIC860 demonstrated activity against FAW in the leaftissue. Further, the chimeric insecticidal protein TIC867 demonstratedactivity against CBW and FAW in the leaf, square and boll tissues, aswell as TBW and SBL in the leaf. The chimeric insecticidal proteinTIC868 demonstrated activity against FAW in the leaf, square and bolltissues, as well as TBW and SBL in the leaf.

TABLE 10 Bioassay activity of chimeric insecticidal proteins from stablytransformed R₁ cotton leaf tissue. CBW FAW TBW SBL Toxin Leaf SquareBoll Leaf Square Boll Leaf Leaf TIC860 + TIC867 + + + + + + + +TIC868 + + + + +

All of the compositions disclosed and claimed herein can be made andexecuted without undue experimentation in light of the presentdisclosure. While the compositions of this invention have been describedin terms of the foregoing illustrative embodiments, it will be apparentto those of skill in the art that variations, changes, modifications,and alterations may be applied to the composition described herein,without departing from the true concept, spirit, and scope of theinvention. More specifically, it will be apparent that certain agentsthat are both chemically and physiologically related may be substitutedfor the agents described herein while the same or similar results wouldbe achieved. All such similar substitutes and modifications apparent tothose skilled in the art are deemed to be within the spirit, scope, andconcept of the invention as defined by the appended claims.

All publications and published patent documents cited in thespecification are incorporated herein by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

What is claimed is:
 1. A chimeric insecticidal protein comprising SEQ IDNO:7, wherein the chimeric insecticidal protein exhibits inhibitoryactivity against an insect species of the order Lepidoptera.
 2. Apolynucleotide encoding the chimeric insecticidal protein of claim 1,wherein the polynucleotide is operably linked to a heterologouspromoter.
 3. A polynucleotide encoding a chimeric insecticidal protein,wherein the polynucleotide: a) comprises SEQ ID NO: 6; or b) encodes thechimeric insecticidal protein of claim
 1. 4. A host cell comprising thepolynucleotide of claim 3, wherein said polynucleotide comprises SEQ IDNO: 6, wherein the host cell is selected from the group consisting of abacterial host cell and a plant host cell.
 5. The host cell of claim 4,wherein the bacterial host cell is selected from the group consisting ofAgrobacterium, Rhizobium, Bacillus, Brevibacillus, Escherichia,Pseudomonas, Klebsiella, and Erwinia.
 6. The host cell of claim 4,wherein said plant host cell is selected from the group of plantsconsisting of monocots and dicots.
 7. An insect inhibitory compositioncomprising the chimeric insecticidal protein of claim
 1. 8. The insectinhibitory composition of claim 7, further comprising at least oneinsect inhibitory agent different from the chimeric insecticidalprotein.
 9. The insect inhibitory composition of claim 8, wherein saidat least one insect inhibitory agent is selected from the groupconsisting of an insect inhibitory protein and an insect inhibitorydsRNA molecule.
 10. The insect inhibitory composition of claim 8,wherein said at least one other pesticidal agent exhibits activityagainst one or more pest species of the orders Lepidoptera, Coleoptera,Hemiptera, Homoptera, or Thysanoptera.
 11. A seed comprising an insectinhibitory effective amount of: a) the chimeric insecticidal protein ofclaim 1; or b) the polynucleotide set forth in SEQ ID NO:
 6. 12. Amethod of controlling a Lepidopteran pest, the method comprisingcontacting the Lepidopteran pest with an inhibitory amount of thechimeric insecticidal protein of claim
 1. 13. A transgenic plant cell,plant or plant part comprising a chimeric insecticidal protein, whereinthe chimeric insecticidal protein comprises SEQ ID NO:
 7. 14. A methodof controlling a Lepidopteran pest, comprising exposing the pest to thetransgenic plant or plant part of claim 13, wherein said plant or plantpart expresses a Lepidopteran inhibitory amount of the chimericinsecticidal protein.
 15. A commodity product derived from the plant orplant part of claim 13, wherein the product comprises a detectableamount of the chimeric insecticidal protein.
 16. The commodity productof claim 15, wherein the product is selected from the group consistingof plant biomass, oil, meal, animal feed, flour, flakes, bran, lint,hulls, and processed seed.
 17. A method of producing a seed comprisingthe chimeric insecticidal protein of claim 1, the method comprising: a)planting at least one seed comprising the chimeric insecticidal proteinof claim 1; b) growing at least one plant from said seed; and c)harvesting seeds from said at least one plant, wherein the harvestedseeds comprise the chimeric insecticidal protein of claim
 1. 18. Arecombinant polynucleotide molecule encoding the chimeric insecticidalprotein of claim 1, said molecule comprising SEQ ID NO:6 and apolynucleotide sequence encoding an insect inhibitory agent differentfrom the chimeric insecticidal protein.
 19. A recombinant nucleic acidmolecule comprising a heterologous promoter operably linked to apolynucleotide segment encoding a chimeric insecticidal protein,wherein: a) in the chimeric insecticidal protein comprises SEQ ID NO: 7;or b) the polynucleotide segment comprises SEQ ID NO: 6; wherein saidchimeric insecticidal protein exhibits inhibitory activity against aninsect species of the order Lepidoptera.